Multiplexed imaging of tissues using mass tags and secondary ion mass spectrometry

ABSTRACT

A method of generating a high resolution two-dimensional image of a sample comprising cells and extracellular structures is provided. In certain embodiments, the method comprises: labeling a sample with at least one mass tag, thereby producing a labeled sample; scanning the sample with a secondary ion mass spectrometer (SIMS) ion beam to generate a data set that comprises spatially-addressable measurements of the abundance of the mass tag across an area of the sample; and outputting the data set. In many embodiments, the data set contains the identity and abundance of the mass tag. A system for performing the method is also provided.

CROSS-REFERENCING

This application claims the benefit of U.S. provisional patentapplication Ser. Nos. 61/877,733, filed on Sep. 13, 2013, and61/970,803, filed on Mar. 26, 2014, which applications are incorporatedby reference in their entireties.

GOVERNMENT RIGHTS

This invention was made with Government support under contract nos.CA034233, AI057229, CA130826, EY018228, HHSN272200700038C, 1K99GM104148-01 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND

Antibodies were first employed in tissue section analysis in 1942 tovisualize pneumococcal antigens in organ biopsies from mice infused withlive bacteria. Since that time, immunohistochemistry (IHC) has become amainstay of clinical diagnostics and basic research that is primarilyused to assess the spatial distribution of one or two (rarely more)antigens in tissue sections. Despite the high specificity of manyantibodies, the concentration of most antigens is insufficient to permitdetection by conventional assays without signal amplification. Signalamplification is typically achieved using multivalent, enzyme-linkedsecondary antibodies that bind the Fc portion of the primary antibody.In bright-field microscopy, the most commonly used enzymatic reporter ishorseradish peroxidase, typically used to oxidize 3,3′-diaminobenzidine(DAB), resulting in accumulation of a brown precipitate. Still, the useof secondary antibodies combined with their poor correlation to theprimary antigen concentration due to non-linear staining limits bothreliable multiplexing and quantitation.

Simultaneous detection of multiple antigens can be subject to additionalconstraints that limit the utility of existing IHC-based analysis forpredictive biomarker development in human clinical trials and clinicaldiagnostics. Colorimetric detection of four antigens has been reportedusing multiple enzyme-linked secondary antibodies, but in practice thisapproach is usually limited to two because of difficulties encounteredin sample preparation and imaging. Fluorescent labels can provide ahigher signal-to-noise ratio and are more frequently used forsimultaneous detection of multiple molecular targets. Practicallimitations include the need for primary antibodies generated indissimilar host species and for non-overlapping reporter emissionspectra. This conventional IHC methodology thus does not support therobust generation of multiplexed, quantitative data needed to understandthe relationship between tissue microarchitecture and expression at amolecular level.

SUMMARY

A method of generating a high resolution two-dimensional image of asample comprising cells and extracellular structures is provided. Incertain embodiments, the method comprises: labeling a sample with atleast one mass tag, thereby producing a labeled sample; scanning thesample with a secondary ion mass spectrometer (SIMS) ion beam togenerate a data set that comprises spatially-addressable measurements ofthe abundance of said mass tag across an area of the sample; andoutputting the data set. In many embodiments, the data set contains theidentity and abundance of the, mass tag.

The sample may be labeled with the at least one mass tag in a variety ofdifferent ways. For example, the mass tag comprised by a histochemicalstain, or it may be conjugated to a capture agent, e.g., an antibody. Inother cases, the sample may have been fed a mass tag while it wasliving, and the mass tag may have been incorporated into other moleculesin the sample by metabolism.

In certain embodiments, the labeling step may involve contacting thesample with at least two different specific binding reagents, eachcomprising a different mass tag. In these embodiments, the sample may bescanned to generate a data set that comprises spatially-addressablemeasurements of the abundance of each of the mass tags across an area ofthe sample.

In particular embodiments, labeling may be done using a specific bindingreagent, e.g., an antibody that contains a chelated atom that functionsas the mass tag, methods for making which are known. In someembodiments, the mass tag may have a mass in the range of 12-238 atomicmass units, e.g., 21 to 238 atomic mass units, including C, O, N and Fadducts. In some embodiments, the mass tag may be an atom of an elementhaving an atomic number in the range of 21-90, e.g., an element havingan atomic number of 21-29, 39-47, 57-79 or 89. In some cases, theelement is a lanthanide.

In some cases, the method may comprise constructing an image of thesample from the data set. In some embodiments, the resolution of theimage may be up to 1 nm, 5 nm, e.g., up to 10 nm, up to 50 nm, up to 100nm, up to 500 nm, or up to 5000 nm.

In some embodiments, the method may further comprise defining theboundaries of individual cells (and, optionally, subcellular features inindividual cells or other features of interest) in the image bysegmenting the image, methods for which are known. In these embodiments,the method may further comprise integrating the data that corresponds toeach the individual cells in the image, or a subcellular featurethereof, which may provide a set of values (each corresponding to anindividual mass tag), that describe the cell as a whole. This embodimentof the method may further comprise categorizing the individual cells(e.g., into cell types or into normal or not normal, etc.) based on theintegrated data obtained for each of the cells. In these embodiments,the method may further comprise displaying an image of the tissuesample, in which the cells are color-coded by their category (i.e.,where the cells of a first category are indicated in a first color, thecells of a second category are indicated in a second color and the cellsof a third category are indicated in a third color, etc.). In somecases, in any one pixel of the image, the intensity of the color of thepixel may correlate with the integrated magnitude of the signalsobtained for that pixel obtained by the scanning.

In some embodiments, the tissue sample is mounted on a conductivesubstrate, and the scanning is done by rastering the ion beam across thesample. The method may be performed on any suitable sample, includingthose from plants, animals, and sections that include microbial, e.g.,bacterial, cells. In particular embodiments, the sample may be a tissuesection, e.g., a formalin-fixed, paraffin-embedded (FFPE) section, thathas a thickness in the range of, e.g., 2 to 20 microns (e.g., 3 to 12microns).

Also provided is a system for analyzing a sample. This system maycomprise: a) a secondary ion mass spectrometry (SIMS) system thatcomprises a holder for retaining a substrate comprising a sample ,wherein the system is configured to (i) scan the sample with a SIMS ionbeam and generate a data set that comprises measurements of theabundance of a specific binding reagent that is bound to the sample and(ii) output the data set; and b) a computer comprising an image analysismodule that processes the data set to produce an image of the sample.The holder is in a movable stage that can be controllably moved (e.g.,stepped or continuously moved) in at least in the x and y directions(which are in the plane of the sample) to facilitate scanning. The imageanalysis module can be programmed to perform many of the steps of themethod described above. For example, in some embodiments, the imageanalysis module may segment the image to identify the boundaries ofindividual cells, and, optionally, subcellular features in individualcells, in the image. In some cases, the image analysis module mayintegrate the data for each of the individual cells or a subcellularfeature thereof in the image and optionally categorize the individualcells based on the integrated data obtained for each of the cells. Theimage analysis module may also displays an image of the tissue sample,wherein the cells are color-coded by their category. As noted above, inany one pixel of the image, the intensity of the color of the pixelcorrelates with the magnitude of the signals obtained for that pixelobtained by the SIMS system.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Certain aspects of the following detailed description are bestunderstood when read in conjunction with the accompanying drawings. Itis emphasized that, according to common practice, the various featuresof the drawings are not to scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1. Workflow summary of multiplexed ion beam imaging (MIBI).Biological specimens, such as FFPE tissue or cell suspensions, areimmobilized on a conductive substrate, such as indium tin oxide coatedglass or silicon wafer. Samples are subsequently stained with antibodiesconjugated to unique transition element isotope reporters, dried, andloaded under vacuum for MIBI analysis. The sample surface is rasterizedwith a primary ion beam (O—) that sputters the antibody-specific isotopereporters native to the sample surface as secondary ions. An elementalmass spectrum is acquired for each pixel. Regions of interestdemarcating nuclear and cytosolic compartments of each cell areintegrated, tabulated, and categorized. Composite images comprised ofpseudo-colored categorical features and quantitative three-coloroverlays are constructed to summarize multidimensional expression data.

FIG. 2. Analysis of PBMCs stained with metal-conjugated antibodies usingmass cytometry and MIBI. (A) PBMCs stained with seven antibodies wereimmobilized on a silicon wafer and imaged using MIBI. Single cellregions of interest were segmented using CD45 surface expression andintegrated for each antibody. (B, C) Hierarchical gating of theresultant data yielded comparable values for seven cell populationsrelative to those found by mass cytometry. (D) Pearson correlation ofthe relative abundance of each cell population demonstrated strongagreement between the two methods (r=0.98, p<0.0001, two-tailed t test).

FIG. 3. 10-color imaging of human breast tumors using MIBI. (A) Avidityof primary antibodies is unaffected by metal-conjugation. To access theeffect of metal conjugation on antibody avidity, immunoperoxidasestaining of serial sections from a single human breast tumor werestained with metal-conjugated or unmodified primary antibodies for Ki67or ER-alpha. Positive-staining nuclei of comparable intensity werepresent in similar numbers when using metal-conjugated or unmodifiedprimary antibodies. (B) Visual representation of MIBI data. Singlechannel ion data can be color mapped and merged to constructpseudo-brightfield or pseudo-darkfield images resembling conventionalimmunoperoxidase or immunofluorescence staining, respectively. (C)10-color imaging of human breast tumors. FFPE tissue sections from threedifferent patients were analyzed using MIBI. HER2, ER, and PR areexpressed appropriately with respect to the known immunophenotype ofeach specimen. ER, PR, and Ki67 demonstrate well-demarcated nuclearpositivity, while e-cadherin, actin, HER2, and keratin expression isappropriately membranous.

FIG. 4. Quantitative analysis of tumor immunophenotype. (A) Forquantitative single cell analysis, ion images are segmented into ROIsdemarcating nuclear and cytoplasmic compartments. (B) Examination of theresultant data using conventional biaxial scatter plots demonstratesquantitative expression patterns matching the known immunophenotype ofeach respective tumor.

FIG. 5. Composite representation of multidimensional MIBI data usingcategorical and quantitative colorization. (A) Quantitative colorizationof cytoplasmic features. Green-encoded e-cadherin, red-encoded actin,and blue-encoded vimentin channels were merged to generate aquantitative representation of protein expression and colocalization.(B) Categorical colorization of nuclei. Subpopulations of ER/PR/Ki67positive or ER/PR positive nuclei are pseudo-colored yellow or aqua,respectively. (C) Multidimensional data are summarized in a compositeimage illustrating quantitative and categorical expression patterns.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

As used herein, the term “labeling” refers to attaching a detectablemoiety to an analyte such that the presence and/or abundance of theanalyte can be determined by evaluating the presence and/or abundance ofthe label. The term “labeling” includes labeling using a histologicalstain (in which case the mass tag may be part of or conjugated to thestain) as well as labeling using a capture agent, e.g., an antibody oran oligonucleotide probe, that has been conjugated to a mass tag. Asample can also be labeled by feeding the sample with a mass-taggedcompound (e.g., IdU or BrdU) that is metabolized and incorporated intothe sample prior to fixation.

As used herein, the term “biological feature of interest” refers to anypart of a cell that can be stained or indicated by binding to anantibody. For example, stains may be used to define and examine bulktissues (highlighting, for example, muscle fibers or connective tissue),cell populations (classifying different blood cells, for instance), ororganelles within is individual cells. Stains may be class-specific(DNA, proteins, lipids, carbohydrates).

Exemplary biological features of interest include cell walls, nuclei,cytoplasm, membrane, keratin, muscle fibers, collagen, bone, proteins,nucleic acid, fat, etc. A biological feature of interest can also beindicated by immunohistological methods, e.g., using a capture agentsuch as an antibody that is conjugated to a label. In these embodiments,the capture agent binds to an epitope, e.g., a protein epitope, in thesample. Exemplary epitopes include, but are not limited tocarcinoembryonic antigen (for identification of adenocarcinomas,cytokeratins (for identification of carcinomas but may also be expressedin some sarcomas) CD15 and CD30 (for Hodgkin's disease), alphafetoprotein (for yolk sac tumors and hepatocellular carcinoma), CD117(for gastrointestinal stromal tumors), CD10 (for renal cell carcinomaand acute lymphoblastic leukemia), prostate specific antigen (forprostate cancer), estrogens and progesterone (for tumouridentification), CD20 (for identification of B-cell lymphomas) and CD3(for identification of T-cell lymphomas).

As used herein, the term “multiplexing” refers to using more than onelabel for the simultaneous or sequential detection and measurement ofbiologically active material.

As used herein, the term “specific binding reagent” refers to a labeledreagent that can specifically bind to one or more sites in a specificmolecular target (e.g., a specific protein, phospholipid, DNA molecule,or RNA molecule) in or on a cell. Specific binding reagents includeantibodies, nucleic acids, and aptamers, for example. A used herein, an“aptamer” is a synthetic oligonucleotide or peptide molecule thatspecifically binds to a specific target molecule.

As used herein, the terms “antibody” and “immunoglobulin” are usedinterchangeably herein and are well understood by those in the field.Those terms refer to a protein consisting of one or more polypeptidesthat specifically binds an antigen. One form of antibody constitutes thebasic structural unit of an antibody. This form is a tetramer andconsists of two identical pairs of antibody chains, each pair having onelight and one heavy chain. In each pair, the light and heavy chainvariable regions are together responsible for binding to an antigen, andthe constant regions are responsible for the antibody effectorfunctions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight is chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,minibodies, single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Alsoencompassed by the term are Fab′, Fv, F(ab′)₂, and or other antibodyfragments that retain specific binding to antigen, and monoclonalantibodies. Antibodies may exist in a variety of other forms including,for example, Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e.bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.Immunol. 17, 105 (1987)) and in single chains (e. g., Huston et al.,Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al.,Science, 242, 423-426 (1988), which are incorporated herein byreference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y.,2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986),).

The term “specific binding” refers to the ability of a binding reagentto preferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between a binding reagent andanalyte when they are specifically bound in a capture agent/analytecomplex is characterized by a K_(D) (dissociation constant) of less than10⁻⁶M, less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than10⁻⁹ M, less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

As used herein, the term “mass tagged” refers to a molecule that istagged with either a single kind of stable isotope that is identifiableby its unique mass or mass profile or a combination of the same, wherethe combination of stable isotopes provides an identifier. Combinationsof stable isotopes permit channel compression and/or barcoding. Examplesof elements that are identifiable by their mass include noble metals andlanthanide, although other elements may be employed. An element mayexist as one or more isotopes, and this term also includes isotopes ofpositively and negatively metals. The terms “mass tagged” and“elementally tagged” may be used interchangeably herein.

As used herein, the term “ mass tag” means any isotope of any element,including transition metals, post transition metals, halides, noblemetal or lanthanide, that is identifiable by its mass, distinguishablefrom other mass tags, and used to tag a biologically active material oranalyte. A mass tag has an atomic mass that is distinguishable from theatomic masses present in the analytical sample and in the particle ofinterest. The term “monoisotopic” means that a tag contains a singletype of metal isotope (although any one tag may contain multiple metalatoms of the same type).

As used herein, the term “lanthanide” means any element having atomicnumbers 58 to 71. Lanthanides are also called “rare earth metals”.

As used herein, the term “noble metal” means any of several metallicelements, the electrochemical potential of which is much more positivethan the potential of the standard hydrogen electrode, therefore, anelement that resists oxidation. Examples include palladium, silver,iridium, platinum and gold.

As used herein, the term “elemental analysis” refers to a method bywhich the presence and/or abundance of elements of a sample areevaluated.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have at least 10, at least 100, at least 100, at least 10,000, atleast 100,000, at least 10⁶, at least 10⁷, at least 10⁸ or at least 10⁹or more members.

A “sample comprising cells” is a sample of biological origin thatcontains intact, e.g., fixed, cells. In some embodiments, the sample maybe substantially planar. Examples of such samples include tissuesections, samples that are made by depositing disassociated cells onto aplanar surface, and samples that are made by growing a sheet of cells ona planar surface.

As used herein, the term “scanning” refers to a method by which a sourceof radiation is (e.g., a laser) is zig-zagged or rastered over a surfaceuntil a substantial two dimensional area has been irradiated by thesource of energy.

As used herein, the term “spatially-addressable measurements” refers toa set of values that are each associated with a specific position on asurface. Spatially-addressable measurements can be mapped to a positionin a sample and can be used to reconstruct an image of the sample.

As used herein, the term “across an area”, in the context ofspatially-addressable measurements of the abundance of a mass tag acrossan area of a sample, refers to measurements of mass tags that are at orunder (e.g., on or within cells that are proximal to) the surface of thesample. The depth of the area analyzed can vary depending on the energyof the ion beam.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

In order to further illustrate the present invention, the followingspecific examples are given with the understanding that they are beingoffered to illustrate the present invention and should not be construedin any way as limiting its scope.

The method described herein employs a mass tag, i.e., a stable isotopethat is identifiable by its mass for labeling of a biological samplethat contains cells and extracellular structures, measured on aninstrument capable of quantifying elemental composition with spatialregistration using a secondary ion mass spectrometer (SIMS), e.g.,static or dynamic SIMS.

The mass tag may be part of or conjugated to a stain, or conjugated to acapture agent such as an antibody. In certain embodiments, mass tags maybe composed of a chelating polymer made up of repeating units of a metalchelator, such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriamine pentaacetic acid (DTPA), chelated to one or more atoms of asingle non-biological isotope. In some embodiments the mass tags may besubstantially is uniform in size, so the abundance of specific bindingreagent will be in direct proportion with the number of tag atoms. Thetagged specific binding reagent is then contacted with a biologicalsample, washed, and measured with an SIMS instrument capable ofquantifying the number of tag atoms present in the sample with spatialregistration. The abundance of the analyte may be inferred from themolar ratio of tag atoms per detection reagent.

The method described above may be multiplexed in that the assay can bedone using multiple specific binding reagents (e.g., more than 2specific binding reagents, up to 5 specific binding reagents, up to 10specific binding reagents, up to 20 specific binding reagents, up to 50specific binding reagents or up to 100 specific binding reagents ormore). Each specific binding reagent may be linked to a different masstag, where the mass tags are distinguishable from one another bysecondary ion mass spectrometry. Alternatively or in addition,multiplexing may involve using stains for specific features of interest.

Many elements exist in nature as multiple stable isotopes. For example,¹⁵³Eu accounts for 52% of europium on Earth and ¹⁵¹Eu makes up most ofthe remaining 48%, while unstable, radioactive isotopes of europiumconstitute less than 1%. Many stable isotopes are commercially availableas powders or salt preparations, in varying degrees of purity, including99% (2N), 99.9% (3N), 99.99% (4N), 99.999% (5N) and 99.9999% (6N) pure.In some embodiments, metal chelator tags may be synthesized usingenriched isotopes. For example, mass dots may be synthesized using 151Eu(e.g. Europium 151 Oxide, 99.999% purity, American Elements). Mass dotsare described in US patent publication 2012/0178183 A1, which isincorporated herein by reference. Using enriched isotopes maximizes thenumber of unique species of isotope tags that can be simultaneouslydetected in a multiplexed analysis. In addition, spatially distinctfeatures of interest may be labeled with the same metal tag to furthermultiplex the analysis. Such spatially distinct features may bedistinguished based on co-localization with one or more other metaltags. For example, a Her2 membrane stain and an ER nuclear stain usingthe same metal tag may be distinguished from one based on a dsDNA orhistone H3 stain that uses a different metal tag, which wouldco-localize with the ER stain.

The mass tag may be part of or conjugated to a stain. In theseembodiments, the stain may be phalloidin, gadodiamide, acridine orange,bismarck brown, barmine, Coomassie blue, bresyl violet, brystal violet,DAPI, hematoxylin, eosin, ethidium bromide, acid fuchsine, haematoxylin,hoechst stains, iodine, malachite green, methyl green, methylene blue,neutral red, Nile blue, Nile red, osmium tetroxide (formal name: osmiumtetraoxide), rhodamine, safranin, phosphotungstic acid, osmiumtetroxide, ruthenium tetroxide, ammonium molybdate, cadmium iodide,carbohydrazide, ferric chloride, hexamine, indium trichloride, lanthanumnitrate, lead acetate, lead citrate, lead(II) nitrate, periodic acid,phosphomolybdic acid, potassium ferricyanide, potassium ferrocyanide,ruthenium red, silver nitrate, silver proteinate, sodium chloroaurate,thallium nitrate, thiosemicarbazide, uranyl acetate, uranyl nitrate,vanadyl sulfate, or any derivative thereof. The stain may be specificfor any feature of interest, such as a protein or class of proteins,phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, an organelle (e.g., cellmembrane, mitochondria, endoplasmic recticulum, golgi body, nulearenvelope, and so forth), a compartment of the cell (e.g., cytosol,nuclear fraction, and so forth). The stain may enhance contrast orimaging of intracellular or extracellular structures.

In certain embodiments, the stain may be suitable for administration toa live subject. The stain may be administered to the subject by anysuitable means, such as ingestion, injection (e.g., into the bloodcirculation), or topical administration (e.g., during a surgery). Such astain may be specific for a tissue, biological structure (e.g., bloodvessel, lesion), or cell type of interest. The stain may be incorporatedinto cells of the subject of a cellular process, such as glucose uptake.Examples of such stains include, without limitation, gadolinium,cisplatin, halogenated carbohydrates (e.g., carbohydrates which arefluorinated, chlorinated, brominated, iodinated), and so forth. Otherinjectable stains used in imaging techniques (e.g., such as MRI, PETscans, CT scans and so forth) may be conjugated to a mass tag if notinherently associated with a mass tag, and administered to a livesubject. A sample may be obtained from the subject after administration,for use in the methods described herein.

In other embodiments, and as will be described in greater detail below,the mass tag may be conjugated to a capture agent, e.g., an antibodythat recognizes an epitope on the sample. In a multiplexed assay, acombination of capture agents and stains may be used.

The mass tag used in the method may be any stable isotope that is notcommonly found in the sample under analysis. These may include, but arenot limited to, the high molecular weight members of the transitionmetals (e.g. Rh, Jr, Cd, Au), post-transition metals (e.g. Al, Ga, In,Tl), metalloids (e.g. Te, Bi), alkaline metals, halogens, and actinides,although others may be used in some circumstances. A mass tag may have amass in the range of 21 to 238 atomic mass units. In certainembodiments, a lanthanide may be use. The lanthanide series of theperiodic table comprises 15 elements, 14 of which have stable isotopes(La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu). Lanthanindescan be readily used because of their rarity in the biosphere. There aregreater than 100, non-biological stable isotopes of elements between 1and 238 AMU . In some embodiments, tagging isotopes may comprisenon-lanthanide elements that can form stable metal chelator tags for theapplications described herein. In SIMS-based measurement modality,unlike some ICP-MS-based modalities, the elemental reporter could alsoconsist of lower MW, transition elements not common in biologicalmatrices (e.g. Al, W, and Hg).

Elements suitable for use in this method in certain embodiments include,but are not limited to, lanthanides and noble metals. In certain cases,an elemental tag may have an atomic number of 21-90. In particularembodiments, the elemental tag may contain a transition metal, i.e., anelement having the following atomic numbers, 21-29, 39-47, 57-79, and89. Transition elements include the lanthanides and noble metals. See,e.g., Cotton and Wilkinson, 1972, pages 528-530. The elemental tagsemployed herein are non-biological in that they are man made and notpresent in typical biological samples, e.g., cells, unless they areprovided exogenously.

In particular embodiments, the mass tag to be linked to the bindingreagent may be of the formula: R-MT, where R is a reactive group thatcan form a linkage with a reactive group on a specific binding reagentand MT is a mass tag. The compound may also contain a spacer between Rand MT. In particular embodiments, R may be, e.g., a maleimide orhalogen-containing group that is sulhydryl reactive, anN-hydroxysuccinimide (NHS)-carbonate that is amine-reactive or anN,N-diisopropyl-2-cyanoethyl phosphoramidite that is hydroxyl-reactive.Such groups react with other groups on the specific binding reagent,e.g., a cysteine or other residue of an antibody or a sulfhydryl groupof an oligonucleotide). In many embodiments, the linkage between thereactive group and the mass tag is not selectively cleavable, e.g., isnot photo-cleavable.

In particular embodiments, MT may be a polymer of, e.g., 10-500 units,where each unit of the polymer contains a coordinated transition metal.Suitable reactive groups and polymers containing coordinating groups,including DOTA and DTPA-based polychetants, are described in a varietyof publications, including: Manabe et al. (Biochemica et Biophysica Acta883: 460-467 (1986)) who describes attaching up to 105 DTPA residuesonto a poly-L-lysine backbone using the cyclic anhydride method and alsoattaching polylysine-poly-DTPA polychelants onto monoclonal antibody(anti-HLA IgG1) using a 2-pyridyl disulphide linker achieving asubstitution of up to about 42.5 chelants (DTPA residues) persite-specific macromolecule; Torchilin (U.S. Pat. No. 6,203,775) whodescribes a generic method for labeling antibodies that includes anantibody-reactive, lanthanide chelating compound of a generic formula;Sieving (U.S. Pat. No. 5,364,614), the abstract for describes aDOTA-based polychetant containing a polylysine backbone that is linkedto a protein. Further descriptions of such moieties are described in,for example: US20080003616 (Polymer backbone element tags), U.S. Pat.No. 6,203,775 (Chelating polymers for labeling of proteins), U.S. Pat.No. 7,267,994 (Element-coded affinity tags), U.S. Pat. No. 6,274,713(Polychelants) and U.S. Pat. No. 5,364,613 (Polychelants containingmacrocyclic chelant moieties), as well as many others. Thesepublications are incorporated by references for their generic andspecific teachings of reactive groups and polymers containingcoordinating groups, as well as the methods that can make suchcompounds. In addition to the methods described in the references citedabove, methods for making polymer-based elemental tags are alsodescribed in detail in Zhang et al (Agnew Chem. Int. Ed. Engl. 2007 46:6111-6114). In addition, any chelator able to bind to metal tags can beused. These include EDTA, EGTA, and Heme. These chelators are able tobind to +1, +2, +3, +4 ions of metal tags. Methods for linking such tagsto binding reagents are known in the art. For example, the MAXPARreagents produced by DVS Sciences is a maleimide-functionalized polymerof DTPA, with an average length of 30 monomers. Using the MAXPARprotocol, it is possible to conjugate a typical IgG antibody with 6 or 7polymers, thereby conjugating an average of 200 tagging isotope atomsper antibody.

When using mass-based elemental analysis there are more than 100non-biological elemental isotopic masses available between 21 and 238atomic mass units (amu) that can be simultaneously measured withvirtually no overlap. Because these rare earth metals are not usuallypresent in biological isolates, the only limitations of detection arethe sensitivity of the reagents to which they are conjugated, and thesensitivity of the instrument performing the measurement.

In particular embodiments, the method described above may be employed ina multiplex assay in which a heterogeneous population of cells islabeled with a plurality of distinguishably mass tagged binding reagents(e.g., a number of different antibodies). As there are more than 80naturally occurring elements having more than 200 stable isotopes, thepopulation of cells may be labeled using at least 5, at least 10, atleast 20, at least 30, at least 50, or at least 100, up to 150 or moredifferent binding reagents (that bind to, for example different cellsurface markers) that are each tagged with a different mass. After thepopulation of cells is labeled, they are analyzed using the methoddescribed above.

As noted above, the specific binding reagent used in the method may beany type of molecule (e.g., an antibody, a peptide-MHC tetramer, anucleic acid (e.g., ssRNA or ssDNA), an aptamer, a ligand specific for acell surface receptor, etc.) that is capable of specific binding to abinding partner in or on cells. The binding partner may be a protein, anucleic acid or another type of cellular macromolecule (e.g., acarbohydrate). The binding partner may be on the cell surface, or it maybe extracellular or intracellular (e.g., associated with the nucleus oranother organelle, or cytoplasmic).

In certain aspects, a specific binding reagent may be an MT conjugatedto a nucleic acid that hybridizes to a specific RNA and/or DNA sequence.The MT conjugated nucleic acid may be used in combination with anysuitable technique for detecting a target (e.g., RNA, DNA, protein orprotein complex), such as standard in-situ hybridization, In-situhybridization utilizing branched DNA probes (e.g., as provided byAffymetrix), proximity ligation (PLA) and rolling circle amplification(e.g., as provided by Olink bioscience), and so forth. In-situhybridization techniques, including those employing branched DNA probesare described by Monya Baker et al. (Nature Methods 9, 787-790 (2012)).Briefly, in-situ hybridization using branched DNA probes utilizes aseries of ssDNA probes, where a first set of DNA probes specificallyhybridizes to the target DNA or RNA sequence, and a second set of DNAprobes may hybridize to a portion of the first set of DNA probes, thusexpanding the number of DNA probes that can bind (indirectly) to asingle DNA or RNA molecule. A third set may bind to the second set ofDNA probes in a likewise manner, and so forth. One or more of the setsof DNA probes may be conjugated to a metal tag to label the target DNAor RNA molecule. Proximity ligation techniques, including detection ofsingle RNA molecules, DNA molecules, and protein complexes are describedby Weibrecht et al. (Nature Methods 9, 787-790 (2012)) which isincorporated herein by reference. Rolling circle amplification isdescribed by Larsson et al. (Nat. Methods 1, 227-232 (2004)), which isincorporated herein by reference. Briefly, in proximity ligationfollowed by rolling circle amplification, a nucleic acid is hybridizedto two proximal RNA or DNA strands, after which the nucleic acid isligated and then amplified, resulting in many copies of the sequencecomplimentary to the nucleic acid. The complimentary sequence istherefore present in higher copy number than the original proximal RNAor DNA strands, and can be more easily detected (e.g., by a MTconjugated nucleic acid that hybridizes to the complimentary sequence).The proximal RNA or DNA stands may each be conjugated to a differentantibody (e.g., where the different antibodies may each be specific fora different protein of a protein complex).

Any of the above techniques may be used to resolve single moleculartargets (e.g., individual RNA molecules, DNA molecules, proteins orprotein complexes). As single molecular targets may be resolvable asdiscrete puncti, a combination of metal isotopes may be used to uniquelylabel the molecular target. In one example, the specific binding reagentmay be a nucleic acid may be conjugated to a unique combination of metalisotopes. In another example, a combination of MT conjugated nucleicacids (e.g., each conjugated to a different mass tag) may be usedtogether to label the molecular target with a unique combination ofmetal isotopes. As such, n number of mass tags could be combinatoriallyused to label 2′ different molecular targets, provided that themolecular targets can be spatially distinguished. The method describedherein may be used to assay a sample of biological origin that containscells, in which the amounts of certain components (e.g., protein,nucleic acid or other molecules) need to be determined. In someembodiments, this analysis may be done using a SIMS instrument (e.g.NanoSIMS by Cameca, NanoTOF by Physical Electronics). Secondary Ion MassSpectrometry is a surface sensitive technique that allows the detectionand localization of the chemical composition of sample surfaces. Theinstrument may use a finely focused, pulsed primary ion beam to desorband ionize molecular species from a sample surface. The resultingsecondary ions are transferred into a mass spectrometer, where they aremass analyzed and quantified using standard mass analyzers (e.g.,time-of-flight, magnetic sector, quadrupole, ion trap, or a combinationsthereof). Displaying the mass spectra that were collected from thesample surface generates chemical images. Each pixel in the resultingessentially represents a mass spectrum. Notably, this instrument wouldonly require ‘unit resolution’—the ability to discriminate massreporters separated by 1 AMU or more. NanoTOF uses a low intensity,pulsed source that is synced with the TOF detector. Cameca uses a DCbeam (i.e. continuous and not pulsed).

When high-speed pulsed or continuous ion beams (primary ions) areirradiated onto the surface of a solid sample at a high vacuum, acomponent of the surface is released into the vacuum by adesorption-ionization phenomenon. The generated positively ornegatively-charged ions (secondary ions) are focused in one direction byan electrical field, and detection is performed at a remote position.When pulsed primary ions are irradiated onto the solid surface,secondary ions having various masses are generated depending on thecomposition of the surface of the sample. Among the secondary ions, anion having a smaller mass flies faster than an ion having a larger massin a TOF tube. Therefore, a measurement of a time between generation anddetection of the secondary ions (flight time) enables the analysis ofmasses of the generated secondary ions to be performed. When primaryions are irradiated, only secondary ions generated at the outermostsurface of a solid sample are released into the vacuum, so thatinformation about the outermost surface (e.g., a depth of less than 1nm, less than 2 nm, less than 5 nm, less than 10 nm, less than 20 nm,less than 50 nm, less than 100 nm, or more than 100 nm) of the samplecan be obtained. In the TOF-SIMS, the amount of irradiated primary ionsis significantly small, so that an organic compound is ionized whilemaintaining its chemical structure, and the structure of the organiccompound can be identified from the mass spectra. The principles ofsecondary ion mass spectrometry are described in, e.g., Belu et al(Biomaterials. 2003 24: 3635-53), Pól et al (Histochem Cell Biol. 2010134: 423-43) and Klitzing (Methods Mol Biol. 2013 950: 483-501).

As noted above, after the initial data is obtained, the data is used toconstruct an image is of the sample. This image may be analyzed toidentify the boundaries of individual cells, and/or subcellular featuresin individual cells, in the image. Computer-implemented methods forsegmenting images of cells are known in the art and range fromrelatively simple thresholding techniques (see, e.g., Korde, et al AnalQuant Cytol Histol. 2009 31, 83-89 and Tuominen et al Breast Cancer Res2010 12, R56), to more sophisticated methods, such as, for instance,adaptive attention windows defined by the maximum cell size (Ko et al. JDigit Imaging 2009 22, 259-274) or gradient flow tracking (Li, et al. JMicrosc 2008 231, 47-58). Some suitable image segmentation methods maybe reviewed in Ko et al (J Digit Imaging. 2009 22: 259-74) and Ong(Comput Biol Med. 1996 26:269-79). Next the data that corresponds toeach of the individual cells, or a subcellular feature thereof, thathave been defined by the segmenting are integrated to provide, for eachcell, values that represent the amount of each of the mass tags withinthe boundary of each cell. This step of the method results in a data setthat contains, for each cell, measurements of the amount of each of themass tags that are associated with the cell. This concept is illustratedin the table shown below.

Tag 1 Tag 2 Tag 3 Tag 4 Tag 5 Cell 1 0.1 0.1 5 3 1 Cell 2 0.2 0.4 4 0.10.1 Cell 3 10 0.1 0.2 0.3 5This data allows one to categorizing the cells in the sample. Forexample, in the example shown in the table above, the three cells arelikely to be different types of cells because they have differentprofiles of mass tags where the profile identifies the category. Inparticular cases, this information may be used to provide a false-colorimage in which each of the cells is color-coded by their category. Assuch, this method may comprise displaying an image of the sample, inwhich the cells are color-coded by their category. In particularembodiments, in any one pixel of the image, the intensity of the colorof the pixel correlates with the magnitude of the signals obtained forthat pixel obtained in the original scanning. In these embodiments, theresulting false color image may show color-code cells in which theintensity of the color in any single pixel of a cell correlates with theamount of specific binding reagent that is associated with thecorresponding area in the sample.

As the original scan may only result in partial removal of the sample(e.g., at a depth on the nanometer scale), the sample may be re-scannedto generate an additional data set having measurements of the abundanceof one or more mass tags across the area that was originally scanned.For example, the original scan may be used to identify an area or areasof interest in the sample. Such a scan may be lower resolution and maytherefore be more rapid, measure the mass tag abundance in a larger areaat a time, and/or may result in removal of less of the sample. There-scan may be a higher resolution scan of the abundance of metal tagsin the area or areas of interest. Alternatively or in addition, multiplescans across the same area may be used to produce a 3 dimensional image(e.g., compiled from the individual 2 dimensional data sets). In certainaspects, areas of interest identified by an original scan may beanalyzed further after isolation of the area of interest from thesample, e.g., such as by laser capture micro dissection.

The methods described herein may include normalization as a means ofstandardizing data obtains across samples and/or time-points (e.g., toenable quantitative cross-sample comparison). In certain aspects,normalization of ionization and/or overall measurement efficiency may beperformed using standardized metal particles or suspension present inthe sample. The standardized metal particles or suspension may have aknown amount of one or more mass tags, and the resulting measurement ofthe one or more mass tags may be used to normalize the measurements ofother mass tags in the sample. For example, normalization beads may beused to calibrate the system or normalize data obtained by the subjectmethods. Normalization of mass cytometry data using bead standards isdescribed by Rachel Fink et al. (Cytometry A. 83(5):483-94(2013)), whichis incorporated herein by reference, and is applicable to the subjectmethods which also utilize time of flight mass spectrometry.Alternatively or in addition, ionization and/or measurement efficiencymay be normalized according to any of the above-mentioned stains. Forexample, measurements of a mass tag used to stain the ER may benormalized to the overall intensity of that mass tag in a given area, inthe cell, or across multiple cells in the sample.

Normalization may also be used to account for the effects of, forexample, degree of tissue fixation, retention of protein, and stainingefficiency with specific binding reagents. Mass tags conjugated towell-characterized antibodies that bind molecular targets stablyexpressed across a wide range of cell types may be used fornormalization. Such antibodies include, without limitation, antibodiesto housekeeping proteins (such as GAPDH, HSP90, beta-actin andbeta-tubulin), dsDNA and histone H3.

As discussed above, the methods of the subject invention allow for amultiplexed approach. Multiple mass tags may be measured to determinethe abundance of multiple molecular targets (e.g. specific proteins,DNA, RNA, etc.) as well as biologic features of interest in the sample(e.g., cell or tissue structure, cellular organelles, cellularfractions, etc.).

In addition, mass tag measurements may be normalized according to any ofthe above-described embodiments. The large number of discrete mass tagsenables multiplexing of more than 2, 5, 10, 20, 30, 40, 50, 60, 70, 80,100 or more different mass tags in a single area. Multiple mass tags(e.g., conjugated to antibodies against complementary epitopes of thesame molecular target) may be used for redundancy so as to increaseconfidence in a measurement of a specific molecular target. Furthermultiplexing may be achieved by using identical mass tags to label twoor more spatially distinct targets or features of interest.Alternatively or in addition, a unique combination of metal tags may beused to identify a spatially distinct target or feature of interest.

Systems

Also provided is a system for analyzing a sample. This system maycomprise: a) a secondary ion mass spectrometry (SIMS) system thatcomprises a holder for retaining a substrate comprising a sample,wherein the system is configured to (i) scan the sample with a primaryion beam (e.g., a beam of oxygen, cesium, gold, argon, bismuth, xenon,C60, SF₆, or gallium ions, or any mixture there, e.g., a mixture ofoxygen and xenon ions) and generate a data set that comprisesmass-specific abundance measurements of a specific binding reagent thatis bound to the sample and (ii) output the data set; and b) a computercomprising an image analysis module that processes the data set toproduce an image of the sample. The holder is in a movable stage thatcan be controllably moved (e.g., stepped or continuously moved) in atleast the x and y directions (which are in the plane of the sample) tofacilitate scanning. The image analysis module can be programmed toperform many of the steps of the method described above. For example, insome embodiments, the image analysis module may segment the image toidentify the boundaries of individual cells, and, optionally,subcellular is features in individual cells, in the image. In somecases, the image analysis module may integrate the data for each of theindividual cells or a subcellular feature thereof in the image andoptionally categorize the individual cells based on the integrated dataobtained for each of the cells. The image analysis module may alsodisplay an image of the tissue sample, wherein the cells and/orsubcellular features thereof are color-coded by their category. As notedabove, in any one pixel of the image, the intensity of the color of thepixel correlates with the magnitude of the signals obtained for thatpixel obtained by the SIMS system. In a particular embodiment, thesystem may comprise a DC ion source (i.e. dynamic source) linked to aquadrapole, then to an ion pulser, then to a time of flight (TOF) tube.The SIMS system may be dynamic or static. NanoSIMS is considered adynamic because it uses a higher power DC primary ion source, NanoTOF isconsidered static because it uses a lower power pulsed source.

The image analysis module may combine data sets obtained from multiplescanned areas into a single data set, wherein each of the multiplescanned areas are offset from one another. The image analysis module mayadjust the offset between adjacent scanned areas so as to increase theoverlap of pixels with similar mass tag intensities near the edges ofthe adjacent scanned areas.

The image analysis module may transform the data set into one or morefalse color images (e.g. pseudocolor, pseudobrightfield,pseudo-immunofluorescence). The image may be in any suitable image fileformat (e.g., JPEG, Exif, TIFF, GIF, PNG, a format readable by an imageanalysis software such as ImageJ, and so forth). In certain embodiments,the image analysis module may produce the image by transforming theabundance (e.g., measured intensity) of one or mass tags into theintensity of one or more false colors at individual pixels in the image.The relationship between the intensity of a mass tag and the intensityof the corresponding false color may be linear or non-linear (e.g.,logarithmic, exponential, etc.).

In certain embodiments, the system is configured to generate amultiplexed data set comprising spatially-addressable measurements ofthe abundances of a plurality of mass tags that are bound to an area ofthe sample. The image analysis module may transform the plurality ofmass tag measurements to produce a plurality of false color images. Theimage is analysis module may overlay the plurality of false color images(e.g., superimpose the false colors at each pixel) to obtain amultiplexed false color image. Multiple mass tag measurements (e.g.,unweighted or weighted) may be transformed into a single false color,e.g., so as to represent a biological feature of interest characterizedby the binding of the specific binding reagent associated with each ofthe multiple mass tags. False colors may be assigned to mass tags orcombinations of mass tags, based on manual input from the user.Alternatively or in addition, an unsupervised approach may be used todetermine groups of mass tags to be represented by a single false color.The unsupervised approach may identify groups of mass tags thatmaximizing variance while minimizing the number of groups (e.g., such asthrough principle component analysis (PCA)), grouping mass tags that areco-localized and/or in proximity (e.g., by any suitable clusteringalgorithm), or may employ any other suitable method for grouping masstags to be represented by a single false color. In certain aspects, theimage may comprise false colors relating only to the intensities of masstags associated with a feature of interest, such as mass tags in thenuclear compartment (e.g., co-localized with a dsDNA specific mass tag).

The image analysis module may further be configured to adjust (e.g.,normalize) the intensity and/or contrast of mass tag intensities orfalse colors, to perform a convolution operation (such as blurring orsharpening of the mass tag intensities or false colors), or perform anyother suitable operations to enhance the image. In certain aspects, theimage analysis module may compile data sets generated from multiple 2Dscans to produce an image that is a 3D model of the cells. The imageanalysis module may perform any of the above operations to align pixelsobtained from successive 2D scans and/or to blur or smooth mass tagintensities or false colors across pixels obtained from successive 2Dscans to produce the 3D model.

The image analysis method may be implemented on a computer. In certainembodiments, a general-purpose computer can be configured to afunctional arrangement for the methods and programs disclosed herein.The hardware architecture of such a computer is well known by a personskilled in the art, and can comprise hardware components including oneor more processors (CPU), a random-access memory (RAM), a read-onlymemory (ROM), an internal or external data storage medium (e.g., harddisk drive). A computer system can also comprise one or more graphicboards for processing and outputting graphical information to displaymeans. The above components can be suitably interconnected via a businside the computer. The computer can further comprise suitableinterfaces for communicating with general-purpose external componentssuch as a monitor, keyboard, mouse, network, etc. In some embodiments,the computer can be capable of parallel processing or can be part of anetwork configured for parallel or distributive computing to increasethe processing power for the present methods and programs. In someembodiments, the program code read out from the storage medium can bewritten into a memory provided in an expanded board inserted in thecomputer, or an expanded unit connected to the computer, and a CPU orthe like provided in the expanded board or expanded unit can actuallyperform a part or all of the operations according to the instructions ofthe program code, so as to accomplish the functions described below. Inother embodiments, the method can be performed using a cloud computingsystem. In these embodiments, the data files and the programming can beexported to a cloud computer, which runs the program, and returns anoutput to the user.

A system can in certain embodiments comprise a computer that includes:a) a central processing unit; b) a main non-volatile storage drive,which can include one or more hard drives, for storing software anddata, where the storage drive is controlled by disk controller; c) asystem memory, e.g., high speed random-access memory (RAM), for storingsystem control programs, data, and application programs, includingprograms and data loaded from non-volatile storage drive; d) systemmemory can also include read-only memory (ROM); a user interface,including one or more input or output devices, such as a mouse, akeypad, and a display; e) an optional network interface card forconnecting to any wired or wireless communication network, e.g., aprinter; and f) an internal bus for interconnecting the aforementionedelements of the system.

The memory of a computer system can be any device that can storeinformation for retrieval by a processor, and can include magnetic oroptical devices, or solid state memory devices (such as volatile ornon-volatile RAM). A memory or memory unit can have more than onephysical memory device of the same or different types (for example, amemory can have multiple memory devices such as multiple drives, cards,or multiple solid state memory devices or some combination of the same).With respect to computer readable media, “permanent memory” refers tomemory that is permanent. Permanent memory is not erased by terminationof the electrical supply to a computer or processor. Computer hard-driveROM (i.e., ROM not used as virtual memory), CD-ROM, floppy disk and DVDare all examples of permanent memory. Random Access Memory (RAM) is anexample of non-permanent (i.e., volatile) memory. A file in permanentmemory can be editable and re-writable.

Operation of computer is controlled primarily by operating system, whichis executed by central processing unit. The operating system can bestored in a system memory. In some embodiments, the operating system canincludes a file system. In addition to an operating system, one possibleimplementation of the system memory includes a variety of programmingfiles and data files for implementing the method described below. Incertain cases, the programming can contain a program, where the programcan be composed of various modules, and a user interface module thatpermits a user at user interface to manually select or change the inputsto or the parameters used by programming. The data files can includevarious inputs for the programming.

In certain embodiments, instructions in accordance with the methoddescribed herein can be coded onto a computer-readable medium in theform of “programming”, where the term “computer readable medium” as usedherein refers to any storage or transmission medium that participates inproviding instructions and/or data to a computer for execution and/orprocessing. Examples of storage media include a floppy disk, hard disk,optical disk, magneto-optical disk, CD-ROM, CD-R, magnetic tape,non-volatile memory card, ROM, DVD-ROM, Blue-ray disk, solid state disk,and network attached storage (NAS), whether or not such devices areinternal or external to the computer. A file containing information canbe “stored” on computer readable medium, where “storing” means recordinginformation such that it is accessible and retrievable at a later dateby a computer.

The computer-implemented method described herein can be executed usingprogramming that can be written in one or more of any number of computerprogramming languages. Such languages include, for example, Java (SunMicrosystems, Inc., Santa Clara, Calif.), Visual Basic (Microsoft Corp.,Redmond, Wash.), and C++(AT&T Corp., Bedminster, N.J.), as well as anymany others.

Utility

The above-described method can be used to analyze a cells from a subjectto determine, for example, whether the cell is normal or not or todetermine whether the cells are responding to a treatment. In oneembodiment, the method may be employed to determine the degree ofdysplasia in cancer cells. In these embodiments, the cells may be from asample of from a multicellular organism or a microbe. A biologicalsample may be isolated from an individual, e.g., from a soft tissue orfrom a bodily fluid, or from a cell culture that is grown in vitro. Abiological sample may be made from a soft tissue such as brain, adrenalgland, skin, lung, spleen, kidney, liver, spleen, lymph node, bonemarrow, bladder stomach, small intestine, large intestine or muscle,etc. Bodily fluids include blood, plasma, saliva, mucous, phlegm,cerebral spinal fluid, pleural fluid, tears, lactal duct fluid, lymph,sputum, cerebrospinal fluid, synovial fluid, urine, amniotic fluid, andsemen, etc. Biological samples also include cells grown in culture invitro. A cell may be a cell of a tissue biopsy, scrape or lavage orcells. In particular embodiments, the cell may of a cell in a formalinfixed paraffin embedded (FFPE) sample. In particular cases, the methodmay be used to distinguish different types of cancer cells in FFPEsamples.

The method described above finds particular utility in examining tissuesections using panels of antibodies, examples of which are provided inthe table below.

Acute Leukemia IHC Panel CD3, CD7, CD20, CD34, CD45, CD56, CD117, MPO,PAX-5, and TdT. Adenocarcinoma vs. Mesothelioma IHC Pan-CK, CEA, MOC-31,BerEP4, TTF1, Panel calretinin, and WT-1. Bladder vs. Prostate CarcinomaIHC Panel CK7, CK20, PSA, CK 903, and p63. Breast IHC Panel ER, PR,Ki-67, and HER2. Reflex to HER2 FISH after HER2 IHC is available.Burkitt vs. DLBC Lymphoma IHC panel BCL-2, c-MYC, Ki-67. CarcinomaUnknown Primary Site, Female CK7, CK20, mammaglobin, ER, TTF1, CEA,(CUPS IHC Panel—Female) CA19-9, S100, synaptophysin, and WT-1. CarcinomaUnknown Primary Site, Male CK7, CK20, TTF1, PSA, CEA, CA19-9, S100,(CUPS IHC Panel—Male) and synaptophysin. GIST IHC Panel CD117, DOG-1,CD34 and desmin. Hepatoma/Cholangio vs. Metastatic HSA (HepPar 1), CDX2,CK7, CK20, CAM 5.2, Carcinoma IHC Panel TTF-1, and CEA (polyclonal).Hodgkin vs. NHL IHC Panel BOB-1, BCL-6, CD3, CD10, CD15, CD20, CD30,CD45 LCA, CD79a, MUM1, OCT-2, PAX-5, and EBER ISH. Lung Cancer IHC Panelchromogranin A, synaptophysin, CK7, p63, and TTF-1. Lung vs. MetastaticBreast Carcinoma TTF1 mammaglobin GCDFP-15 (BRST-2), and IHC Panel ER.Lymphoma Phenotype IHC Panel BCL-2, BCL-6, CD3, CD4, CD5, CD7, CD8,CD10, CD15, CD20, CD30, CD79a, CD138, cyclin D1, Ki67, MUM1, PAX-5, TdT,and EBER ISH. Lymphoma vs. Carcinoma IHC Panel CD30, CD45, CD68, CD117,pan-keratin, MPO, S100, and synaptophysin. Lymphoma vs. ReactiveHyperplasia IHC BCL-2, BCL-6, CD3, CD5, CD10, CD20, CD23, Panel CD43,cyclin D1, and Ki-67. Melanoma vs. Squamous Cell Carcinoma CD68, FactorXIIIa, CEA (polyclonal), S-100, IHC Panel melanoma cocktail (HMB-45,MART-1/Melan-A, tyrosinase) and Pan-CK. Mismatch Repair Proteins IHCPanel MLH1, MSH2, MSH6, and PMS2. (MMR/Colon Cancer) NeuroendocrineNeoplasm IHC Panel CD56, synaptophysin, chromogranin A, TTF-1, Pan-CK,and CEA (polyclonal). Plasma Cell Neoplasm IHC Panel CD19, CD20, CD38,CD43, CD56, CD79a, CD138, cyclin D1, EMA, kappa, lambda, and MUM1.Prostate vs. Colon Carcinoma IHC Panel CDX2, CK 20, CEA (monoclonal)CA19-9, PLAP, CK 7, and PSA. Soft Tissue Tumor IHC Panel Pan-CK, SMA,desmin, S100, CD34, vimentin, and CD68. T-Cell Lymphoma IHC panel ALK1,CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD20, CD21, CD30, CD56, TdT, andEBER ISH. T-LGL Leukemia IHC panel CD3, CD8, granzyme B, and TIA-1.Undifferentiated Tumor IHC Panel Pan-CK, S100, CD45, and vimentin.

In some embodiments, the method may involve obtaining an image asdescribed above (an electronic form of which may have been forwardedfrom a remote location) and may be analyzed by a doctor or other medicalprofessional to determine whether a patient has abnormal cells (e.g.,cancerous cells) or which type of abnormal cells are present. The imagemay be used as a diagnostic to determine whether the subject has adisease or condition, e.g., a cancer. In certain embodiments, the methodmay be used to determine the stage of a cancer, to identify metastasizedcells, or to monitor a patient's response to a treatment, for example.

In any embodiment, data can be forwarded to a “remote location”, where“remote location,” means a location other than the location at which theimage is examined. For example, a remote location could be anotherlocation (e.g., office, lab, etc.) in the same city, another location ina different city, another location in a different state, anotherlocation in a different country, etc. As such, when one item isindicated as being “remote” from another, what is meant is that the twoitems can be in the same room but separated, or at least in differentrooms or different buildings, and can be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” informationreferences transmitting the data representing that information aselectrical signals over a suitable communication channel (e.g., aprivate or public network). “Forwarding” an item refers to any means ofgetting that item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. Examples of communicatingmedia include radio or infra-red transmission channels as well as anetwork connection to another computer or networked device, and theinternet or including email transmissions and information recorded onwebsites and the like. In certain embodiments, the image may be analyzedby an MD or other qualified medical professional, and a report based onthe results of the analysis of the image may be forwarded to the patientfrom which the sample was obtained.

In some cases, the method may be employed in a variety of diagnostic,drug discovery, and research applications that include, but are notlimited to, diagnosis or monitoring of a disease or condition (where theimage identifies a marker for the disease or condition), discovery ofdrug targets (where the a marker in the image may be targeted for drugtherapy), drug screening (where the effects of a drug are monitored by amarker shown in the image), determining drug susceptibility (where drugsusceptibility is associated with a marker) and basic research (where isit desirable to measure the differences between cells in a sample).

In certain embodiments, two different samples may be compared using theabove methods. The different samples may be composed of an“experimental” sample, i.e., a sample of interest, and a “control”sample to which the experimental sample may be compared. In manyembodiments, the different samples are pairs of cell types or fractionsthereof, one cell type being a cell type of interest, e.g., an abnormalcell, and the other a control, e.g., normal, cell. If two fractions ofcells are compared, the fractions are usually the same fraction fromeach of the two cells. In certain embodiments, however, two fractions ofthe same cell may be compared. Exemplary cell type pairs include, forexample, cells isolated from a tissue biopsy (e.g., from a tissue havinga disease such as colon, breast, prostate, lung, skin cancer, orinfected with a pathogen etc.) and normal cells from the same tissue,usually from the same patient; cells grown in tissue culture that areimmortal (e.g., cells with a proliferative mutation or an immortalizingtransgene), infected with a pathogen, or treated (e.g., withenvironmental or chemical agents such as peptides, hormones, alteredtemperature, growth condition, physical stress, cellular transformation,etc.), and a normal cell (e.g., a cell that is otherwise identical tothe experimental cell except that it is not immortal, infected, ortreated, etc.); a cell isolated from a mammal with a cancer, a disease,a geriatric mammal, or a mammal exposed to a condition, and a cell froma mammal of the same species, preferably from the same family, that ishealthy or young; and differentiated cells and non-differentiated cellsfrom the same mammal (e.g., one cell being the progenitor of the otherin a mammal, for example). In one embodiment, cells of different types,e.g., neuronal and non-neuronal cells, or cells of different status(e.g., before and after a stimulus on the cells) may be employed. Inanother embodiment of the invention, the experimental material is cellssusceptible to infection by a pathogen such as a virus, e.g., humanimmunodeficiency virus (HIV), etc., and the control material is cellsresistant to infection by the pathogen. In another embodiment, thesample pair is represented by undifferentiated cells, e.g., stem cells,and differentiated cells. Cells any organism, e.g., from bacteria,yeast, plants and animals, such as fish, birds, reptiles, amphibians andmammals may be used in the subject methods. In certain embodiments,mammalian cells, i.e., cells from mice, rabbits, primates, or humans, orcultured derivatives thereof, may be used.

In order to further illustrate the present invention, the followingspecific examples are given with the understanding that they are beingoffered to illustrate the present invention and should not be construedin any way as limiting its scope.

EXAMPLES

Described below is an example of a method that uses secondary ion massspectrometry to image antibodies via isotopically pure elemental metalreporters. Multiplexed ion beam imaging (MIBI) is capable of analyzingup to 100 or more targets simultaneously with 50-nm lateral resolutionover a five log dynamic range. Here, MIBI is used to analyzeformalin-fixed, paraffin-embedded (FFPE) human breast tumor tissuesections using 10 labels simultaneously. The resultant data suggest MIBIwill provide new insights relating tissue microarchitecture and highlymultiplexed protein expression patterns relevant to clinicaldiagnostics, basic research, and drug discovery.

This method circumvents some of the limitations associated withconventional light based staining methods. This method can be used onvirtually any vacuum-compatible specimen, including FFPE tissue. Invalidating this method we were able to demonstrate an almostquantitatively identical immunophenotypic analysis of PBMCs compared toa more conventional approach (FIG. 2) as well as equivalent (stainingpattern and intensity) imaging of three FFPE breast tumors withdifferent immunophenotypes with the additional benefit of ten or moremarkers being analyzed simultaneously (FIG. 3). Additionally, markermultiplexing and image segmentation permitted quantitative featureextraction describing cellular and subcellular expression, that inaggregate, revealed immunophenotypes of cell subpopulations that couldbe related back to the original clinical pathology of the tissue (FIG.4). Finally, novel approaches in combinatorial false coloring (or pseudocoloring) of images could distill the high dimensional analysis down toa rapidly interpretable single image where multiple phenotypes could berepresented by a single color in an unsupervised fashion (FIG. 5). Suchrepresentations create an opportunity for vastly improvingmicroscopy-based diagnostics by leveraging readily obtainable andinterpretable high dimensional information using MIBI-like approaches.

MIBI has advantages over conventional IHC techniques. Background signaldue to autofluorescence is absent and the dynamic range presented hereis already 10⁵, exceeding immunofluorescence and chromogenic IHC by100-fold and 1000-fold, respectively. Because the mass resolution isless than one hundredth of a dalton, no spectral overlap is observedbetween different metal-conjugated primary antibodies, obviating theneed for channel compensation. Assay linearity is improved relative toboth chromogenic IHC and IF because neither secondary labeling noramplified detection are required. Meanwhile, relatively conventionalmethods are used for immunoreactions, and because mass tags do notdegrade, samples are stable indefinitely, permitting remote preparationtogether with a centralized reading facility.

Reagents can be developed which extends the capability of MIBI to otherarenas and away from antibody-based analysis, such as in situhybridization and subcellular metabolic analysis. Taken together, theextended capabilities of MIBI permitted by relatively minormodifications of existing analytical systems introduce the prospect of apractical, multiplexed imaging platform that integrates tissuehistology, protein expression, gene expression, and metabolism on asubcellular level.

METHODS

Substrate preparation: Silicon wafers (Silicon Valley Microelectronics)were diced into 18 mm² pieces, rinsed two times with methanol, andpolished with a cotton-tipped applicator. Cleaned substrates weresubsequently immersed in 2% poly-1-lysine solution (Sigma-Aldrich) for10 min and baked at 60° C. for 1 hr.

Antibodies: A summary of antibodies, reporter isotopes, andconcentrations can be found in table 51 below. Metal conjugated primaryantibodies were prepared 100m at a time using the MaxPAR antibodyconjugation kit (DVS Sciences, Toronto, Canada) according to themanufacturer's recommended protocol. Following labeling, antibodies werediluted in Candor PBS Antibody Stabilization solution (Candor BioscienceGmbH, Wangen, Germany) to 0.4 mg/mL and stored long-term at 4° C.

TABLE S1 Antigen Vendor Clone Mass Element PBMC Primary Antibodies CD45Biolegend HI30 115 In CD19 DVS Sciences HIB19 142 Nd CD4 DVS SciencesRPA-T4 145 Nd CD14 DVS Sciences M5E2 160 Gd CD8 Biolegnd RPA-T8 165 HoCD3 DVS Sciences UCHT1 170 Er HLA-DR DVS Sciences L243 174 Yb BreastTumor Primary Antibodies ER alpha Labvision 1D5 139 La PR cellsignalD8Q2J 145 Nd Ki67 Labvision Polyclonal 150 Nd Vimentin Cellsignal D21H3154 Sm E-cadherin Cellsignal 24E10 158 Gd Pan-keratin Cellsignal C11 162Dy Her2 Cellsignal D8F12 166 Er Pan-actin Cellsignal D18C11 168 Er dsDNAAbeam HYB331-01 176 Yb

Cells: Unmatched human peripheral blood was purchased from the StanfordBlood Bank according to an IRB-approved protocol. All blood samples werecollected in heparin sulfate anticoagulant, stored at room temperaturefor 4-6 hrs, and then separated over Ficoll-Paque Plus (AmershamBiosciences) using Accuspin tubes (Sigma-Aldrich, St. Louis, Mo.) toremove erythrocytes, platelets, and granulocytes. Cells were frozen inFCS with 10% DMSO. Cells were rested at 37° C., 5% CO2 for 1 hour inRPMI with 10% FCS (supplemented with 2mM EDTA in the case of frozensamples), 1× L-glutamine and 1× penicillin/streptomycin (Invitrogen).

Staining of peripheral blood mononuclear cells: Cellular stainingprotocols were based on procedures previously described. Briefly, afterresting cells for 1 hr, surface marker antibodies were added yielding100 μL final reaction volumes and incubated at room temperature for 30min. Following incubation, cells were washed two times with cellstaining media and split into two aliquots. For mass cytometry analysis,cells were permeabilized with 4° C. methanol for 10 min at 4° C., washedtwice with cell staining media to remove residual methanol, and thenstained with 1 mL of 1:4000 191/193Ir DNA intercalator diluted in PBSwith 1.6% PFA for 20 mins at room temperature. Cells were then washedonce with cell staining media, once with PBS, and then diluted in dH2Oto approximately 106 cells/mL prior to analysis. For MIBI analysis, 50μL of cells diluted in PBS to approximately 10⁷ cells/mL were placed onsilicon substrate and allowed to adhere for 20 min. The substrate wasthen gently rinsed with PBS, fixed for 5 min in PBS with 2%glutaraldehyde, and rinsed twice with dH2O. Lastly, samples weredehydrated via a graded ethanol series, air dried at room temperature,and stored in a vacuum desiccator for at least 24 hrs prior to analysis.

Breast tumor tissue sections: Tissue sections (4 μm thickness) were cutfrom FFPE tissue blocks of human breast tumor using a microtome, mountedon poly-1-lysine-coated silicon substrate for MIBI analysis or a glassslide for immunoperoxidase (IPDX) staining. Silicon-mounted sectionswere baked at 65° C. for 15 min, deparaffinized in xylene, andrehydrated via a graded ethanol series. The sections were then immersedin epitope retrieval buffer (10 mM sodium citrate, pH 6) and placed in apressure cooker for 30 min (Electron Microscopy Sciences, Hatfield,Pa.). The sections were subsequently rinsed twice with dH₂O and oncewith wash buffer (TBS, 0.1% Tween, pH 7.2). Residual buffer was removedby gently touching the surface with a lint-free tissue prior toincubating with blocking buffer for 30 min (TBS, 0.1% Tween, 3% BSA, 10%donkey serum, pH 7.2). Blocking buffer was subsequently removed and thesections were stained overnight at 4° C. in a humidified chamber. Thefollowing morning, the sections were rinsed twice in wash buffer,postfixed for 5 min (PBS, 2% glutaraldehyde), rinsed in dH₂O, andstained with Harris hematoxylin for 10 s. Finally, the sections weredehydrated via graded ethanol series, air dried at room temperature, andthen stored in a vacuum desiccator for at least 24 hrs prior to imaging.Antigen retrieval was performed using a Decloaking Chamber (BiocareMedical, Concord, Calif.) with citrate buffer at pH 6.0, 125° C. andpressure to 15 psi. The total time slides were in the chamber was 45min. Incubations with primary antibodies were performed at roomtemperature overnight in a humidified chamber. Normal goat serum wasused for blocking. Biotinylated goat anti-rabbit (1:1000) was thesecondary antibody used with a Vectastain ABC Kit Elite and a PeroxidaseSubstrate Kit DAB (Vector Labs, Burlingame, Calif.) used foramplification and visualization of signal, respectively. Tissues knownto contain each assessed antigen were used as positive controls.

MIBI analysis: MIBI analysis was performed with a NanoSIMS 50L massspectrometer (Cameca) using an O-primary ion beam supplied by an oxygenduoplasmatron source. The primary optics, secondary optics, and massspectrometer were tuned prior to each experiment. The seven detectortrolleys were calibrated using metal conjugated antibody standardsimplanted on silicon. The detector trolleys were first moved along thefocal plane to the mass peak of the metal corresponding to eachantibody. Mass peaks were then centered on the detectors by performinghigh mass resolution (HMR) scans and adjusting the deflector voltage ofeach trolley. Horizontal and vertical beam alignment was tuned tomaximize secondary ion transmission through the entrance slit of themass spectrometer. Then, the z-position of the sample stage was adjustedsuch that the secondary ion signal was maximal when the voltage of thethird electrode of the immersion lens (E0S) was approximately 7150 V.The primary ion beam was centered on the region of interest (ROI) bytuning lenses Lduo, L0, and L1. All data were taken in positive ion modeusing D1 aperture 2, DO aperture 0 or 3, L1 voltage of approximately1500 V, entrance slit 0, and aperture slit 0. Images containing morethan seven channels were acquired by recalibrating the detector trolleysbetween repeat scans of the same field. ROIs identified on serialsections using brightfield microscopy were located using the CCD camerain the NanoSIMS analysis chamber. Samples were implanted with O-at highprimary ion current until the secondary ion yield had reached steadystate. The Oct-90 and Oct-45 voltages of the stigmator octopole weremanually adjusted to minimize image distortion while viewing a realtimeion image (RTI) of a periodic aluminum grid. Prior to each imageacquisition, the field of view was manually focused by adjusting thevoltage of the second electrode of the immersion lens (EOP) whileviewing a RTI. Ion images were acquired over a 50-100 μm fields of viewwith pixel dwell times between 2-10 ms and up to 10 repeat scans over asingle area. Total scan time for a single field of view ranged between5-25 min. Larger areas were constructed by stitching together multiplecontiguous fields of view into a single mosaic.

Mass cytometry measurement: Cell events were collected on a CyTOF masscytometer as previously described. With detection in dual counting modeusing the ‘data’ calibration, cell length was set to range from 10 to 75with a convolution threshold of 100. A detector stability delay of 20seconds was used and all samples were diluted such that the acquisitionrate was less than 500 cells per second.

PBMC mosaic stitching: The MIBI PBMC data was collected in a series of1200 individual square 50 μm (128 pixel) tiles, arranged in a 40×30rectangle. The relative positions of the tiles were determined using thelog-transformed CD45 images. The reported offset between adjacent tileswas 40 μm in both the x- and y-directions, but the actual offset wasobserved to vary due to imprecision in the stage's location. To accountfor this, each tile was initially placed according to its reportedoffset, and then moved around 1-20 pixels in both the x- andy-directions to multiple different positions. At each location, thecorrelation in the overlap area between the new tile and previous tilewas computed. The tile was then assigned to the position that maximizedthe correlation of the overlapped areas.

PBMC image segmentation: The log-transformed mosaic of CD45 tiles wasconvolved with a 2-dimensional Gaussian kernel with standard deviationof 3 pixels, and then thresholded at a density of 1. Each continuousregion with density greater than this threshold was preliminarilylabeled as an individual cell. The next step was to separate into theirconstituent singlets any sets of multiple cells that were close enoughto be initially labeled as single cells. To do this, for eachpreliminary cell, the two points on the boundary were identified betweenwhich there was the maximum ratio of distance along the boundary toEuclidean distance (the “pinch points”). When this ratio exceeded 0.42(a heuristic cutoff), the preliminary cell was separated into two cellswith a new border segment between the pinch points. This process wasiterated over all cells, and repeated with each new preliminary cellcreated, until no cells had pinch points that exceeded this separationcriteria.

Once the cell boundaries were determined, the raw values of each channelmeasured were summed within each boundary to create a table of total ionintensity on a per-cell basis. The number of pixels within each cell wasalso calculated as a measure of cell size. This table was equivalent toan .fcs file such as from a standard mass cytometry experiment.

Data analysis: To filter out doublets and debris, singlets were gatedfrom the mass cytometry PBMCs by applying standard cell-length by DNAand then cell-length by CD45 gates; a singlet gate using cell area byCD45 was applied to the MIBI PBMCs. The subsequent gating scheme forboth the MIBI and CyTOF processed PBMCs is shown in FIGS. 2B and C,respectively.

Results

Performance assessment of MIBI: The workflow for MIBI is comparable toimmunofluorescence (IF) and chromogenic IHC assays (FIG. 1). Instead offluorophores or enzyme-conjugated reagents, biological specimens areincubated with primary antibodies coupled to isotopically pure, stablelanthanides (FIG. 1). Primary antibodies are combined in solution forsimultaneous incubation with the specimen. The specimens prepared forMIBI are mounted in a sample holder and subjected to a rasterized oxygenduoplasmatron primary ion beam. The impact of this ion beam on thesample liberates lanthanide adducts of the bound antibodies as secondaryions. In this study, the secondary ions are subsequently analyzed via amagnetic sector mass spectrometer equipped with multiple photomultipliertubes, permitting parallel detection of multiple lanthanide isotopes(mass-based reporters). The resultant data produces a two-dimensionalmap of the elemental distribution of each lanthanide, and thus eachantibody and its corresponding epitope.

Peripheral blood mononuclear cells (PBMC) stained with seven metalisotopeconjugated primary antibodies (CD3, CD4, CD8, CD14, CD19, CD45,HLA-DR) were assessed in parallel using mass cytometry and MIBI (FIG.2). Mass cytometry was performed on the PBMC suspension as describedpreviously. For MIBI, cells were immobilized on a poly-1-lysine-coatedsilicon wafer, dried under vacuum, and subsequently analyzed using aNanoSIMS 50L™ mass spectrometer. Sequential 50-□m fields were imaged andconstructed into a composite mosaic for each antibody (FIG. 2A). Theresultant mosaic was segmented into single-cell regions of interest(ROIs) using the CD45 channel. To extract single cell expression datafor each antibody, the ion count for each channel was integrated foreach cell ROI.

Mass cytometry and MIBI produced comparable results and qualitativepatterns of expression when analyzed via traditional biaxial plots (FIG.2B) with marker intensity of MIBI having a dynamic range of 105.Additionally, both platforms yielded quantitatively similar frequenciesfor seven manually gated cell populations (FIG. 2C), with three of thesepopulations differing by less than 1% between platforms (B-cells, CD8+T-cells, CD4+ T-cells). Altogether, using PBMCs as a test case, MIBIcould yield both qualitatively and quantitatively equivalent results asa conventional analytical platform with the additional benefit ofspatial information.

Ten-color imaging of human breast tumor tissue sections: In order toutilize MIBI for analysis of tissue sections acquired in a diagnosticsetting, we sought to verify the activity of metal-conjugated reagentsused in conventional IHC staining by comparing metal-conjugated andunmodified primary antibodies. Secondary staining of serial sectionsfrom a single FFPE human breast tumor tissue block treated withmetal-conjugated or unmodified primary antibodies for Ki67 or estrogenreceptor alpha (ER) demonstrate positive nuclear staining of comparableintensity and similar levels of background staining (FIG. 3A),indicating that the metal conjugation did not materially affect specificand non-specific staining behavior.

Finally, to assess the overall performance of MIBI in a diagnosticimaging application FFPE breast tumor tissue sections from threedifferent patients were analyzed. ER, progesterone receptor (PR), andHER2 positivity were verified in a clinical IHC lab using validatedreagents. For MIBI, tumor sections were mounted on poly-1-lysine-coatedsilicon wafers, deparaffinized, and subjected to heat-induced epitoperetrieval prior to overnight staining with metal-conjugated antibodiesfor dsDNA, ER, progesterone receptor (PR), e-cadherin, Ki67, vimentin,actin, keratin, and HER2. Conveniently, a hematoxylin counterstain canbe readily detected by measuring its elemental aluminum content. Thefollowing day the sections were washed, counterstained with hematoxylin,and dehydrated via graded ethanol series.

Using the MIBI analysis, conventional high resolution images can beconstructed of FFPE tissues. Pseudo-brightfield images mimickingtraditional DAB staining were constructed by encoding hematoxylin on awhite to blue scale while putting the desired marker on a white to brownscale (FIG. 3B, top). Pseudo-fluorescence images mimicking three-colorimmunofluorescence were constructed using a red-encoded dsDNA channel, ablue-encoded hematoxylin channel, and a green encoded marker channel(FIG. 3B, bottom). Pseudo-brightfield and pseudo-fluorescence compositesfor each antibody within a single field of view are shown for each ofthe three tissue sections in FIG. 3C. Comparison of HER2, ER, and PRpositivity across the three specimens demonstrates appropriateexpression with respect to immunophenotypes established by conventionalIHC staining. Sections expressing ER and PR demonstrate well-demarcatednuclear staining, scattered Ki67-positive nuclei, and intense positivestaining for vimentin in mesenchymal cells. HER2-positive sectionsdemonstrate strong membrane staining. Ecadherin, actin, and keratin alsodemonstrate appropriate subcellular staining patterns.

Image segmentation and feature extraction from simultaneously acquiredmarkers: In order to fully leverage the nature of the informationinherent in the quantitatively multiplexed images in this study, imagesegmentation was performed so that cellular features could be analyzedand compared. Hematoxylin and dsDNA channels for each tumor weresegmented using CellProfiler in order to extract summary statisticsdescribing subcellular expression (FIG. 4A). Mean pixel intensities werequantified for each marker within nuclear cytoplasmic, and cellular ROIsfor each cell. Biaxial scatter plots demonstrate marker coexpressionmatching the known immunophenotype for each tumor (FIG. 4B).Triple-positive and ER-PR doublepositive tumors demonstrate nuclearco-expression of ER and PR that is absent in the HER2-positive tumor.Triple-positive and HER2-positive tumors demonstrate cytoplasmicHER2-positivity that is absent in the ER-PR double-positive tumor.Subpopulations of keratin, e-cadherin-positive ductal cells aredistinctly segregated from vimentin-positive mesenchymal cells.

Integrated histological and immunophenotypic features ofmultidimensional MIBI data can be visualized by generating compositeimages that combine quantitative (continuous) cytoplasmic andcategorical (positive or negative) nuclear expression patterns (FIG. 5).Hormone-receptor-positive regions within the epithelial compartment,showing variable non-nuclear expression of actin (red) and e-cadherin(green), can be distinguished from interspersed mesenchymal cellsco-expressing actin (red) and vimentin (blue). Approximately 8% of cellsare seen to be Ki67-positive. Unlike conventional chromogenic IHC, whichis not well-suited to detecting colocalization of multiple markers, MIBIanalysis readily demonstrates ER-PR doublepositive (aqua) or ER-PR-Ki67triple-positive (yellow) subpopulations. In this instance, low-abundanceproliferating cell populations co-expressed ER and PR. It is conceivablethat detailed, cell-by-cell, analysis of molecular phenotypes,especially if multiple nuclear antigen expression profiles are queried,may prove to have practical clinical significance, identifying subsetsof malignant cells that may have different responses to therapy than thebulk tumor population. These observations, combined with thequantitative dynamic range of mass spectrometry approaches like MIBIlends itself to potential diagnostic applications where suchco-localizations and interactions may now be identified in anunsupervised fashion.

1. A method of generating a high resolution two-dimensional image of asample comprising cells and extracellular structures, the methodcomprising: labeling a sample with at least one mass tag, therebyproducing a labeled sample in which a biological feature of interest isbound to said at least one mass tag; scanning the sample with asecondary ion mass spectrometer (SIMS) ion beam to generate a data setthat comprises spatially-addressable measurements of the abundance ofsaid at least one mass tag across an area of said sample; and outputtingthe data set. 2.-24. (canceled)